Abstract/Summary The mammalian palate is anatomically divided into the anterior bony hard palate and the posterior muscular soft palate. However, how the anterior hard palate is patterned and how the palatal osteogenesis is controlled remain unknown. It was recently demonstrated that in the developing branchial arches, the TALE superclass homeodomain proteins particularly Meis proteins set up a ground state that is common to all the arches and their derivatives whereas Hox transcription factors act as tissue-specific cofactor to specify the arch identity. However, this raises a fundamental question as what factors interact with TALE factors to specify and pattern the Hox-free first arch and its derivatives including the palate. The homeobox gene Shox2 is expressed specifically in the anterior palatal mesenchyme, overlapping with the future bony hard palate domain. We have shown previously that Shox2 mutation leads to not only a rare type anterior clefting of the secondary palate, but also the significantly reduced bone formation in the hard palate, which, together with -/- the virtual loss of the stylopod in Shox2 limb, indicates an essential role for Shox2 in organ patterning and skeletogenesis. Our preliminary studies present evidence that Shox2 mutation leads to premature/ectopic + expression of Runx2 in Shox2-expressing palatal cells. RNA-Seq on Shox2 cells from E13 palatal shelves and limbs demonstrates a genome-wide elevated expression of osteogenic genes in the absence of Shox2, consistent with the observation that Shox2 overexpression in cranial neural crest lineage cells inhibits osteogenesis. Moreover, Shox2 ChIP-Seq on the developing palate and limb reveals genome-wide preferential occupation of Shox2 on the responsive cis-regulatory elements of genes bound by Hox and TALE proteins, suggesting that in the Hox-free developing palate, Shox2 functions together with TALE factors to pattern the hard palate and regulates osteogenesis. Based on the abovementioned observations, we hypothesize that in the Hox-free palatal shelf, Shox2 interacts with TALE factors to establish the hard palate identity by antagonizing Meis transcriptional output to prevent premature osteogenesis. In this application, three specific aims are proposed to test this novel hypothesis rigorously: 1) to test the hypothesis that Shox2 expression prevents premature osteogenesis in the developing palate; 2) to test the hypothesis that Shox2 antagonizes the transcription output of Meis and to establish the functional mechanisms for Shox2; 3) to determine the tissue specific chromatin landscape and identify specific enhancer elements underlying palate formation. The results obtained will provide novel knowledge for understanding of palate development and cleft palate formation, and provide solid foundation for future tissue engineering of palatal bone for clinical treatment/repair of cleft palate defects.